The goal of this proposal is to engineer enzyme candidates suitable for the treatment of Celiac Sprue. Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten in genetically susceptible individuals. By damaging the intestinal villi, Celiac Sprue causes a range of symptoms, including diarrhea, iron and vitamin deficiencies, reduced bone density, stunted growth, fatigue, anemia and osteoporosis, increased risks of intestinal cancer and lymphomas. The only treatment for the disease currently available is complete avoidance of gluten, which is very difficult because gluten proteins are present in almost all grains. Proteolytically resistant proline-rich peptides from gluten have been implicated as the immunotoxic agent for Celiac patients. Preliminary results have indicated that bacterial prolyl endopeptidases can degrade these peptides and may thus be an effective treatment for celiac patients. However the naturally occurring endopeptidases all have shortcomings that prevent their therapeutic application. The aim of this project is to use a sequence-activity based protein engineering technology to optimize the activities of prolyl endopeptidases for treatment of Celiac Sprue. Enzymes will be obtained with increased activity towards a full spectrum of gluten-derived proteolytically-resistant peptides, and optimal pH profiles for oral administration. Briefly the experimental methods are as follows. (1) Use phylogenetic and structural modeling to identify amino acid substitutions likely to improve these desired enzyme properties. (2) Design and synthesize specific enzyme variants. (3) Use HPLC to accurately measure the ability of each variant to degrade gluten and its immunotoxic peptides under appropriate conditions. (4) Use the results from activity testing to derive sequence-activity relationships and thus design further improved variants.